The present invention relates to insecticidal microbes with enhanced insecticidal activity. More particularly, the present invention relates to insecticidal baculoviruses in which a genetic sequence coding for an insect selective toxin has been introduced into the genome of the baculovirus.
Venom is defined as a mixture of substances which are produced in specialized glandular tissues in the body of a venomous animal. The venom may be introduced into the body of its prey or opponent, such as by the aid of a stinging-piercing apparatus, in order to paralyze and/or kill it, although other means of delivering venom are known. Scorpions, for example, contain in their venom a number of proteins, or neurotoxins, which are toxic and act on the nervous system. The individual neurotoxins differ in their potency on various species of animals.
The venoms derived from scorpions belonging to the Buthinae subfamily have three main groups of polypeptide neurotoxins which modify axonal sodium conductance. One group of neurotoxins are the xcex1-toxins, which specifically affect mammals through an extreme prolongation of the action potentials due to a slowing or blockage of the sodium channel in activation (Catterall, Science, 223:653-661 (1984); Rochat et al., Advances in Cytopharmacology, pp. 325-334 (1979)). The second group of neurotoxins are the depressant insect selective toxins which induce a progressively developing flaccid paralysis of insects by the blockage of action potentials substantially due to the suppression of sodium current (Lester et al., Biochim. Biophys. Acta, 701:370-381 (1982); Zlotkin et al., Arch. Biochem. Biophys., 240:877-887 (1985)). The third group of neurotoxins are the excitatory insect selective toxins which cause an immediate (knock down) spastic paralysis of insects by the induction of repetitive firing in their motor nerves due to an increase of the sodium peak current and the voltage dependent slowing of its inactivation (Walther et al., J. Insect Physiol., 22:1187-1194; Pelhate et al., J. Physiol., 30:318-319 (1981)).
The scorpion venom derived insect toxins were detected and their isolation was monitored by the typical responses of Sarcophaga blowfly larva which develop an immediate and transient contraction paralysis when injected with the excitatory toxins and progressively developing flaccidity for the depressant toxins (Zlotkin et al., Toxicon, 9:1-8 (1971); Lester et al., Biochim. Biophys. Acta., 701:370-381 (1982)). In spite of the opposite symptomatology induced by the depressant and excitatory insect toxins, both affect exclusively insect sodium channels and share the same binding site (Zlotkin et al., Arch. Biochem. Biophys. 240:877-887 (1985); Gordon et al., Biochim. Biophys. Acta, 778:349-358 (1984)).
Insect-selective toxins have also been identified in venoms from snails, spiders, and a number of other arthropods. [See review by Zlotkin, Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 10, Chapter 15, pp. 499-541 (1985).] The venoms of braconid wasps are highly toxic to lepidopterous larvae. The venom of the braconid Bracon hebetor causes a flaccid paralysis in lepidopterous larvae by inducing presynaptic interruption of the excitatory glutaminergic transmission at the insect neuromuscular junction (Piek et al., Comp. Biochem. Physiol., 72C:303-309 (1982)). The venoms of solitary wasps are toxic to a large number of insects and spiders from different orders (Rathmeyer, Z. Vergl. Physiol., 45:453-462 (1962)). An example of these venoms is the venom of Philanthus triangulum which induces in insects a flaccid paralysis substantially due to presynaptic blockage of neuromuscular transmission; this venom affects both excitatory and inhibitory transmission (May et al., Insect Physiol., 25:285-691 (1979)). The venom of the black widow spider, Latrodectus mactans, contains components which are neurotoxic to insects, but not to mammals, and other components with the opposite selectivity (Fritz et al., Nature, 238:486-487 (1980); Ornberg et al., Toxicon, 14:329-333 (1976)).
Polyhedrosis viruses have been identified as potentially useful to express foreign genes in cells. Smith et al., U.S. Pat. No. 4,745,051, discloses a method for producing a recombinant baculovirus expression vector capable of expression of a selected gene in a host insect cell utilizing baculoviruses from Autographa californica, Trichoplusia ni, Rachiplusia ou, and Galleria mellonella. Bishop, Trends in Biotech., vol. 3 (1988), relates to genetic engineering of baculoviruses for improved pesticidal activity. At page 3, Bishop discusses the possibility of including foreign genes in a nuclear polyhedrosis virus of the alfalfa looper moth Autographa californica. Fraser et al., U.S. Pat. No. 4,870,023, relates to recombinant Autographa californica and Heliothis zea baculoviruses which encode fusion polyhedrin proteins capable of forming occlusion bodies containing foreign peptides. At column 40, Fraser et al. suggest that genes coding for neurotoxins may be expressed in order to increase the insecticidal activity of baculoviruses.
Maeda et al., European Patent Application, publication number 0 222 412, discloses methods of producing insulin-like growth factor I (IGF-1) using recombinant Bombyx mori nuclear polyhedrosis viruses. Maeda, in Invertebrate Cell System Applications, Vol. I (J. Mitsuhashi, ed., CRC Press, Boca Raton, Fla., 1990) describes gene transfer vectors of the Bombyx mori nuclear polyhedrosis virus, and their use for expression of foreign genes in insect cells. Adachi et al., J. Biol. Chem., 264:7681-7685 (1989), relates to the cDNA structure of the bombyxin protein. Maeda, European Patent Application, publication number 0 225 777, with a corresponding U.S. patent issued Nov. 30, 1993 as U.S. Pat. No. 5,266,314, relates to recombinant viruses containing DNA segments of two different nuclear polyhedrosis viruses from different host insects. The recombinant virus is disclosed by Maeda to be effective in destroying or controlling both species of host insects. Maeda, Biochem. and Biophys. Research Communications, 165:1177-1183 (1989) discloses that genetically engineered insect viruses containing a recombinant gene encoding the diuretic hormone of the tobacco hornworm resulted in increased insecticidal activity of the baculovirus.
Miller et al., Science, 219:715-721 (1983) contains the suggestion that recombinant DNA technology could be used to enhance the toxicity of a virus, for example by introducing an insect-specific toxin gene into the genome of Autographa californica nuclear polyhedrosis virus.
Until the present invention, however, attempts to express genes coding for insect-selective neurotoxins and thereby increase the insecticidal activity of recombinant baculoviruses have been unsuccessful. Carbonell et al., Gene, 73:409-418 (1988) attempted to use insect-specific scorpion neurotoxins to improve the effectiveness of baculovirus pesticides. Carbonell et al. cloned the gene encoding insectotoxin-1 of the scorpion Buthus eupeus in E. coli, and attempted to express the gene in Autographa californica nuclear polyhedrosis virus. However, Carbonell et al. reported that biological activity of the toxin was not observed, and no paralytic activity was detected. Carbonell et al., Applied and Environmental Microbiology, 53:1412-1417 (1987) further report that insect baculoviruses did not express genes in infected mammalian cell lines. Dee et al., Bio/Technology, 8:339-342 (1990) report the expression of insecticidal neurotoxin AaIT under the control of a Moloney murine sarcoma virus promoter in cultured mouse cells. However, Dee et al. report that they were unsuccessful in expressing the neurotoxin in E. coli. 
Thus, the identification of a suitable foreign gene and an appropriate signal sequence that are effective for insect control is crucial for the construction of a baculovirus insecticide.
In the present invention, the disadvantages of the prior art in being unsuccessful in expressing scorpion neurotoxins in baculoviruses have largely been overcome.
Accordingly, it is one object of the present invention to provide baculoviruses with enhanced insecticidal activity.
It is another object of the present invention to provide expression systems to produce insect-selective scorpion neurotoxins.
The present invention is directed to insecticidal baculoviruses which possess a genetically enhanced toxicity to insects through the introduction of genes which will induce the production of insect-selective toxins, such as those normally found in scorpions, by the baculovirus. This invention further relates to methods of enhancing the toxicity of the insecticidal baculoviruses comprising incorporating into the genome of the baculovirus a recombinant DNA molecule comprising a genetic sequence coding for a toxin selective for insects.
For example, the toxicity of the Bombyx mori nuclear polyhedrosis virus may be enhanced by introducing a recombinant. DNA molecule coding for an insect toxin. The coding genetic sequence may be operably linked to a secretion signal sequence in order to aid the toxicity. The signal sequence may be synthesized based on the sequence of an insect protein, for example, the signal sequence of the Bombyx mori protein bombyxin.